GBE Evolutionary Relationships among Extinct and Extant Sloths: The Evidence of Mitogenomes and Retroviruses Graham J. Slater1,2,y,PinCui3,y,Analı´aM.Forasiepi4,DorinaLenz3, Kyriakos Tsangaras3, Bryson Voirin5, NadiadeMoraes-Barros6, Ross D. E. MacPhee7,*, and Alex D. Greenwood3,8,* 1Department of Paleobiology & Division of Mammals, National Museum of Natural History, Smithsonian Institution, Washington, DC 2Department of the Geophysical Sciences, University of Chicago 3Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany 4CCT-Mendoza, IANIGLA, Mendoza, Argentina 5Max Planck Institute for Ornithology, Seewiesen, Germany 6Cibio/Inbio - Centro De Investigac¸a˜o Em Biodiversidade E Recursos Gene´ticos, Universidade Do Porto, Vaira˜o, Portugal 7Department of Mammalogy and Division of Vertebrate Zoology, American Museum of Natural History, New York, NY 8Department of Veterinary Medicine, Freie Universita¨t Berlin, Berlin, Germany yThese authors contributed equally to this work. *Corresponding author: E-mail: [email protected];[email protected]. Accepted: February 5, 2016 Data deposition: This project has been deposited at GenBank under the accession KR336791-KR336795 and KR703280-KR703472 and the Sequence Read Archive under the accession SRR2007671-SRR2007675. Abstract Macroevolutionary trends exhibited by retroviruses are complex and not entirely understood. The sloth endogenized foamy-like retrovirus (SloEFV), which demonstrates incongruence in virus–host evolution among extant sloths (Order Folivora), has not been investigated heretofore in any extinct sloth lineages and its premodern history within folivorans is therefore unknown. Determining retroviral coevolutionary trends requires a robust phylogeny of the viral host, but the highly reduced modern sloth fauna (6 species in 2 genera) does not adequately represent what was once a highly diversified clade (~100 genera) of placental mammals. At present, the amount of molecular data available for extinct sloth taxa is limited, and analytical results based on these data tend to conflict with phylogenetic inferences made on the basis of morphological studies. To augment the molecular data set, we applied hybridization capture and next-generation Illumina sequencing to two extinct and three extant sloth species to retrieve full mitochondrial genomes (mitogenomes) from the hosts and the polymerase gene of SloEFV. The results produced a fully resolved and well-supported phylogeny that sup- ports dividing crown families into two major clades: 1) The three-toed sloth, Bradypus, and Nothrotheriidae and 2) Megalonychidae, including the two-toed sloth, Choloepus, and Mylodontidae. Our calibrated time tree indicates that the Miocene epoch (23.5 Ma), particularly its earlier part, was an important interval for folivoran diversification. Both extant and extinct sloths demonstrate multiple complex invasions of SloEFV into the ancestral sloth germline followed by subsequent introgressions across different sloth lineages. Thus, sloth mitogenome and SloEFV evolution occurred sepa- rately and in parallel among sloths. Key words: sloth, mitogenomes, foamy virus, retrovirus–host coevolution, ancient DNA, hybridization capture. Introduction which together form the sister grouptoCingulata(armadillos Superorder Xenarthra comprises one of the most remarkable of and relatives). Although still an important part of the South the major extant groups of placental mammals. Its constituent American mammal fauna, much about their phylogenetic history orders include Folivora (sloths) and Vermilingua (anteaters), remains profoundly obscure (Murphy et al. 2001; Meredith et al. ß The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Genome Biol. Evol. 8(3):607–621. doi:10.1093/gbe/evw023 Advance Access publication February 14, 2016 607 Slater et al. GBE FIG.1.—Constraint tree used for divergence time estimation in BEAST under the fossilized birth-death process. Extinct taxa are depicted at the midpoints of their stratigraphic ranges (supplementary table S2, Supplementary Material online) though analyses integrate over uncertainty regarding true ages. Partitioning of the tree into named taxonomic groups at the family and higher levels is provided for general reference. 2011; DosReisetal.2012; O’Leary et al. 2013). Endogenous (Katzourakis et al. 2009), before the estimated divergence retroviruses (ERVs) represent ancient viral infections that have of two- and three-toed sloths (~21 Ma) but subsequent to become integrated into a host germline and are thereafter trans- the anteater-sloth divergence (~55 Ma). This inference is of mitted vertically as simple Mendelian traits. Widespread ERVs interest because sloths comprise a formerly diverse group of within a taxon may be considered to be fixed. ERVs evolve neu- placental mammals, with nearly 100 species described in the trally (Kijima and Innan 2009; Feschotte and Gilbert 2012), at fossil record (McKenna and Bell 1997; fig. 1). Although clearly rates much slower than those of their exogenous counterparts. an ancient group, they were mostly limited to South America Optimally, rates exhibited by retroviral loci and those of the host until mid- to late Cenozoic times, when certain lineages germline will be in essential synchrony, thus permitting their po- reached various islands in the West Indies and North tential use as codivergence markers for phylogenetic studies. A America (Steadman et al. 2005; MacPhee et al. 2007; group of ERVs known as foamy viruses are widely distributed Gaudin and McDonald 2008; McDonald and De Iuliis 2008). among eutherian mammals and appear to have undergone Paleontologically, the oldest reasonably well preserved fossils coevolution and cospeciation in synchrony with their hosts for assigned to Folivora are Oligocene (Carlini and Scillato-Yane´ more than 100 Myr (Katzourakis et al. 2014). Foamy viruses 2004; Pujos and De Iuliis 2007), although it must be empha- are nonpathogenic, complex retroviruses and form a unique sized that better support is needed to test whether these ear- group among the Retroviridae, Spumaretrovirinae (Linial 1999). liest sloths actually fit cladistically among the crown group (see A recent study of ERVs in extant sloths concluded that sloth Discussion). Declining post-Miocene diversity eventually culmi- endogenized foamy virus (SloEFV) invaded the genome of a nated during the late Quaternary in the loss of all sloth taxa, sloth ancestor in the late Middle Eocene some 39 Ma except for the surviving tree sloths: The three-toed Bradypus 608 Genome Biol. Evol. 8(3):607–621. doi:10.1093/gbe/evw023 Advance Access publication February 14, 2016 Evolutionary Relationships among Extinct and Extant Sloths GBE (Bradypus tridactylus, Bradypus variegatus, Bradypus torqua- et al. 2013, 2015) as well as among closely related mammals tus,andBradypus pygmaeus)andthetwo-toedCholoepus (Switzer et al. 2005; Jern et al. 2006; Katzourakis et al. 2014). (Choloepus hoffmanni and Choloepus didactylus)sloth In consequence, phylogenetic dissociation between host and species. ERVs certainly occurs, but detecting it from patterns of infec- Given how restricted the extant complement of sloths is to tion or introgression in extant taxa can be difficult because of make any headway in understanding the evolution of ERVs the long time spans involved and because many hosts and within folivorans, it would be highly desirable to investigate exogenous retroviral counterparts within taxonomic groups their incidence in extinct forms. Initial attempts to obtain an- are extinct. However, when retroviral and host evolution are cient DNA (aDNA) sequences from different species of Late synchronous, retroviruses can be useful in determining host Pleistocene terrestrial (“ground”) sloths for phylogenetic pur- phylogenetic relationships to the study of cross-species trans- poses were broadly successful (Ho¨ ss et al. 1996; Poinar et al. mission and retroviral introgression patterns require a well- 1998, 2003; Greenwood et al. 2000; Hofreiteretal.2003; resolved host phylogeny. Clack et al. 2012), suggesting that it may be possible to obtain sequence information on SloEFVs occurring in extinct species. Samples A barrier to better understanding of SloEFV evolution within An ~13,000-year-old Mylodon darwinii bone specimen (pre- this group remains because morphological (Gaudin 1995, viously described in Greenwood et al. 2000)andaMylodon 2004; White and MacPhee 2001) and previous molecular coprolite sample (age not determined) were obtained from analyses of sloth phylogeny have failed to reach a consensus the Natural History Museum, London (sample number on higher level relationships among extinct and extant taxa. BM(NH)M8758) (table 1). Two ~20,000-year-old coprolite To clarify phylogenetic relationships and estimate diver- samples attributed to Nothrotheriops shastensis, one from gence dates among and between sloth lineages, as well as Gypsum (USNM 617523), Nevada, and the other from to determine SloEFV macroevolutionary patterns within foli- Rampart Cave (USNM 617524), Arizona, were provided by vorans, we determined complete mitogenomes